The fidelity of cell culture simulations of traumatic brain injury (TBI) that yield tolerance and mechanistic information relies on both the cellular models and mechanical insult parameters. We have designed and characterized an electro-mechanical cell shearing device in order to produce a controlled high strain rate injury (up to 0.50 strain, 30 s−1 strain rate) that deforms three-dimensional (3-D) neural cultures (neurons or astrocytes in an extracellular matrix scaffold). Theoretical analysis revealed that these parameters generate a heterogeneous 3-D strain field throughout the cultures that is dependent on initial cell orientation within the matrix, resulting in various combinations of normal and shear strain. The ability to create a linear shear strain field over a range of input parameters was verified by tracking fluorescent microbeads in an acellular matrix during maximal displacement for a range of strains and strain rates. In addition, cell death was demonstrated in rat cortical astrocytes and neurons in response to high rate, high magnitude shear strain. Furthermore, cell response within the 3-D neuronal cultures depended on orientation, with higher predicted shear strain correlating with an increased loss of neurites, indicating that culture configuration may be an important factor in the mechanical, and hence cellular, response to traumatic insults. Collectively, these results suggest that differential responses exist within a 3-D culture subjected to mechanical insult, perhaps mimicking the in vivo environment, and that this new model can be used to investigate the complex cellular mechanisms associated with TBI.